Developmental changes in rat sciatic nerve myelin.
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Biomedical subjects
Publications and source records attributed to C Linington.
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Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small-sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M-sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate-labelled lectins (WGA, Con A, RCA-60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.
Centrifugation of adult rabbit sciatic nerve homogenates on continuous sucrose density gradients yields three distinct maxima at 0.12, 0.33 and 0.57 M sucrose (labeled A, B and C), respectively. Fraction A is absent in homogenates of immature rabbit sciatic nerve and the material isolated from adult animals is found to contain large amounts of triglycerides. It is proposed that fraction A results from the presence of adipose tissue adhering to the adult nerve preparations and should not be regarded as a true myelin subfraction. The concentration of the major membrane lipid classes (phospholipid, cholesterol and galactosyl ceramide) decreases from the light to the heavy side of the gradient. The ethanolamine phosphatide to sphingomyelin ratio increases from 1.06 to 1.42 between fractions B and C, due to an increase in the phosphatidal ethanolamine content at the expense of sphingomyelin.
The suitability of isolated central nerve myelin preparations for probe labelling studies was assessed and the accessibility of galactosyl ceramides in myelin to galactose oxidase and sodium periodate was determined. Isolated myelin preparations present a uniform external membrane surface to added probes because lamellae in the myelin sheath separate at their external apposition surfaces exclusively during isolation. The cytoplasmic apposition remains intact in isolated myelin. Cationised ferritin can gain access along external apposition regions of inner lamellae in multilamellar fragments of isolated myelin, indicating that proteins and lipids on the external membrane surface will be accessible to probes. Over 50% of the total galactosyl ceramides of myelin are accessible to galactose oxidase attack; hydroxy fatty acid- and nonhydroxy fatty acid-containing cerebrosides are equally attacked. Sodium periodate attacks over 90% of the galactosyl ceramides in isolated myelin at 20 degrees C and electron micrographs of the periodate-treated myelin reveal changes at the external apposition only. Galactosyl ceramides in vesicles of myelin lipid vesicles are not so readily attacked by periodate. The disposition of galactosyl ceramides in the myelin lamellae is discussed.
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Cerebrosides are concentrated in the myelin sheath where they account for about 20% of the total lipid of the membrane. The present paper is concerned with the role and localisation of these glycolipids in the myelin lamellae. Isolated central nerve myelin preparations have been treated with two probes to investigate cerebroside accessibility in the membrane. The action of galactose oxidase on the galactose headgroups of cerebrosides is followed and quantitated by recovery of the modified glycolipid and resolution of either the 6-aldehydo sugar or galactose remaining by gas-liquid chromatography. With isolated myelin preparations only some 40--50% of the cerebroside galactose is attacked by galactose oxidase at 20 degrees C. With periodate at 20 degrees C over 90% of the galactose headgroups are oxidised in 3 h while the figure is 50--55% over the same time period at 4 degrees C rising to 85% after 22 h. With multilamellar liposomes of mixed myelin lipids only some 20--25% of the available cerebroside is oxidised at 4 degrees C, the reaction being complete in 2 h. The results are discussed in relation to the dispositon of cerebroside in the myelin lamellae. A major location on the external face of the membrane system (intraperiod dense line) is favoured. A role for cerebroside in myelin in terms of increasing the stability and resistance of the lipid phase to ion movement is suggested.
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